KR102432567B1 - Mutant fusion protein of nipah virus and its use - Google Patents

Abstract

The present invention relates to a fusion mutant protein derived from Nipah virus and uses thereof. The Nipah virus fusion mutant protein of the present invention has improved expression of the Nipah virus fusion protein, which is known to have a very low expression level, and can be effectively used in the development of a therapeutic agent for Nipah virus infection.

Description

Nipah virus fusion mutant protein and use thereof

The present invention relates to a fusion mutant protein derived from Nipah virus and uses thereof. The Nipah virus fusion mutant protein of the present invention has improved expression of the Nipah virus fusion protein, which is known to have a very low expression level, and can be effectively used in the development of a therapeutic agent for Nipah virus infection.

Nipah virus (NiV) is an RNA virus belonging to the genus henipavirus of the paramyxoviridae family as a zoonotic virus originating in the Asia-Pacific region. Nipah virus has a negative-strand RNA of 18.2 kb as a gene, and these genes are nucleocapisd (N), phosphoprotein (P), matrix (M), fusion (F), attachment glycoprotein (G), large polymerase (L). ) is composed of Nipah virus exhibits a wide host affinity and must be handled in a BSL-4 facility due to its high pathogenicity and mortality.

Nipah virus is a new strain of virus that infects pigs from fruit bats and spreads to humans or other animals that come into direct contact with secretions and discharges such as nasal mucus and saliva of pigs, causing acute and febrile diseases. Starting with the death of 105 people infected with Nipah virus in a pig farm in Malaysia in late 1998, several deaths have been reported in Singapore and southern India, and recently, human-to-human transmission has also been reported. In humans, the incubation period is 418 days, and at the beginning of the onset of symptoms, flu-like fever and muscle pain are shown, but when severe, severe pneumonia and encephalitis can lead to death. It is known that the fatality rate is very high at 70%. The natural host, fruit bat, has no clinical symptoms, but the intermediate host, pig, shows mainly respiratory and neurological symptoms. Like other paramyxoviruses, Nipah virus is known to infect by attachment of an attachment glycoprotein (G) and a fusion protein (F) to a receptor of a host cell.

Currently, there is no effective vaccine or treatment for Nipah virus, and its diagnosis relies on reverse transcriptase polymerase chain reaction (RT-PCR) of viral RNA and immunohistochemical analysis of infected tissues. have. Therefore, development of vaccines, therapeutics, and safer and simpler diagnostic methods is required, and although the introduction of Nipah virus has not been reported in Korea yet, it is a high-risk pathogen, so it is necessary to prepare for the introduction.

With respect to the increase in protein expression, it has been reported that the protein expression level is greatly improved by the substitution of proline in the loop between the HR and the central helix of the MERS virus surface protein (Non-Patent Document 1). This is to increase the expression level by substituting a proline residue for an amino acid present in a loop in which a conformational change occurs significantly when membrane fusion occurs.

In order to develop a vaccine or therapeutic agent for Nipah virus, it is very important to secure a Nipah virus fusion protein. However, it is known that the expression of the Nipah virus fusion protein is very low, and studies on increasing the expression level by substituting amino acid residues of the Nipah virus fusion protein have not been conducted. Accordingly, the present inventors tried to improve the expression rate of Nipah virus fusion protein through genetic manipulation.

Non-Patent Document 1: Pallesen J et al., Proc Natl Acad Sci US A. 2017;114(35):E7348-e57.

An object of the present invention is to provide a fusion protein that can be effectively used in the development of a therapeutic agent for Nipah virus infection by improving the expression rate of the Nipah virus fusion protein, which has a very low expression level.

To this end, the present invention provides a Nipah virus fusion mutant protein having an improved expression rate through genetic manipulation.

In order to solve the above problems, the present invention provides a Nipah virus fusion mutant protein having an improved expression rate.

Specifically, the present invention provides a Nipah virus fusion mutant protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

The present invention also provides a gene encoding the Nipah virus fusion mutant protein, an expression vector comprising the gene, and a host cell transfected with the expression vector.

The present invention also provides the steps of preparing an expression vector comprising a gene encoding the Nipah virus fusion mutant protein, transfecting the expression vector into a host cell, culturing the transfected host cell to produce a virus , It provides a method for producing a mutant Nipah virus fusion protein comprising the steps of infecting and culturing the virus to obtain a culture, and isolating and purifying the protein from the culture.

In the present invention, the host cells may be insect cells, preferably Sf-9 ( Spodoptera frugiperda 9 ) insect cells.

The present invention also provides a vaccine composition for the prevention or treatment of Nipah virus infection comprising the Nipah virus fusion mutant protein as an active ingredient.

In addition, the present invention provides a method for preventing or treating a nipavirus infection comprising administering the vaccine composition to an animal.

The Nipah virus fusion mutant protein according to the present invention has improved expression rate and structural stability of the Nipah virus fusion protein, and can be utilized to develop vaccines and therapeutic agents for prevention or treatment of Nipah virus infection.

In addition, the Nipah virus fusion mutant protein of the present invention is a recombinant protein antigen that can be rapidly produced, and can be applied to the development of a diagnostic agent based on a protein antigen.

1 and 2 show the structure of the Nipah virus fusion protein.
3 to 6 show the purification results of the Nipah virus fusion double mutant (I190P/I114N) protein.
7 to 10 are results of confirming the expression of Nipah virus fusion protein or fusion mutant protein.

Hereinafter, with reference to the accompanying drawings, embodiments and examples of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily carry out. However, the present application may be embodied in various forms and is not limited to the embodiments and examples described herein.

Throughout this specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a Nipah virus fusion mutant protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

Throughout this specification, I190P mutant protein, I114N mutant protein and double mutant (I190P/I114N) protein refer to Nipah virus fusion mutant protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

The present invention also relates to a gene encoding the Nipah virus fusion mutant protein, an expression vector comprising the gene, and a host cell transfected with the expression vector.

As used herein, the term "expression vector" refers to a preparation containing a nucleic acid sequence operatively linked to suitable regulatory sequences capable of expressing a gene in a suitable host cell.

As used herein, the term “operatively linked” means that the binding between nucleic acid sequences is functionally linked.

As used herein, the term “transfection” refers to a change in the genetic properties of a host cell by introducing a foreign gene into a host cell.

As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell in which a gene introduced into the cell is linked to a cellular gene so that it can be replicated and transformed by the cell's own mechanism.

The present invention also provides the steps of preparing an expression vector comprising a gene encoding the Nipah virus fusion mutant protein, transfecting the expression vector into a host cell, culturing the transfected host cell to produce a virus , It relates to a method for producing a Nipah virus fusion mutant protein comprising the steps of infecting and culturing the virus to obtain a culture, and isolating and purifying the protein from the culture.

In one embodiment of the invention, the host cell may be an insect cell. Preferably, the host cell may be an Sf-9 ( Spodoptera frugiperda 9 ) insect cell.

The present invention also relates to a vaccine composition for preventing or treating Nipah virus infection comprising the Nipah virus fusion mutant protein as an active ingredient. In addition, the present invention relates to a method for preventing or treating a nipavirus infection comprising administering the vaccine composition to an animal.

As used herein, the term “vaccine” refers to prevention of infection or re-infection of a corresponding pathogen by inducing an immune response against the pathogen in an animal, including a human, as a host, reducing the severity of symptoms or eliminating symptoms caused by the pathogen, or the pathogen. It is meant to include the substantial or complete elimination of a disease caused by the pathogen. Therefore, the "vaccine composition" of the present invention can be administered to animals, including humans, prophylactically before infection with the pathogen or therapeutically after infection with the pathogen.

As used herein, the term “immune response” is meant to include a humoral immune response, a cellular immune response, or both.

As used herein, the term “active ingredient” refers to a component capable of inducing an immune response by itself or in combination with a carrier that cannot induce an immune response by itself. Therefore, the active ingredient itself does not necessarily have immunogenicity (ability to induce an immune response). In the present invention, the active ingredient is not essential, but is preferably purified.

As used herein, the term "purified" refers to a state in which components other than the target material, such as cellular components of the organism in which the target material is present, are substantially reduced or removed, and the purity is 50% or more, preferably 90%. or more, more preferably 99% or more. Purity may be evaluated through methods known in the art, such as chromatography, gel electrophoresis, immunological analysis, and the like, and purification methods may also use methods known in the art.

The vaccine composition of the present invention may be prepared in any suitable, pharmaceutically acceptable formulation. For example, it may be in the form of a solution or suspension for immediate administration, a concentrated stock solution suitable for dilution prior to administration, or may be prepared in a reconstituted form such as a freeze-dried, freeze-dried, or frozen formulation.

The vaccine composition of the present invention may be formulated with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier usually includes a diluent, excipient, stabilizer, preservative, and the like. For example, as a diluent that may be included in the vaccine composition of the present invention, a non-aqueous solvent such as vegetable oil such as propylene glycol, polyethylene glycol, olive oil, peanut oil, or an aqueous solvent such as water including saline, buffer medium, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, Glycol, water, ethanol, etc. are mentioned, and the stabilizer is carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose; protein. Examples of the preservative include thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, and the like.

The vaccine composition of the present invention may further comprise an antigen adjuvant. Antigen adjuvant is one or more substances that enhance an immune response to an antigen, for example, complete Freund, incomplete Freund, saponin, aluminum adjuvant in gel form, surface active substances (eg, lysolecithin, plurone) Glycol, polyanion, peptide, oil or hydrocarbon emulsion, etc.), vegetable oil (cottonseed oil, peanut oil, corn oil, etc.), may be at least one selected from the group consisting of vitamin E acetate, but is not limited thereto.

The vaccine composition of the present invention may be administered parenterally or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, etc. The range varies accordingly. In addition, the prophylactically or therapeutically effective amount of the composition may vary depending on the administration method, the target site, and the condition of the patient, and when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency.

The present invention will be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example 1] Structural analysis of Nipah virus fusion protein

To model the structure of the Nipah virus fusion protein, the structure of the envelope antigenic surface protein with a trimer structure among respiratory virus proteins was analyzed.

Sequence alignment was performed using MUSCLE and Clustal omega, Geneious programs. In addition, the PDB ID 5EVM structure deposited in the PDB (protein databank) was analyzed, and the structure of the Nipah virus fusion protein was modeled in a triplex form using the PyMOL program, and is shown in FIGS. 1 and 2 .

1 is a schematic diagram of the primary structure of a Nipah virus fusion protein (FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; F1 and F2 are two subunits cleaved by a host protease). Figure 2 is a structure (PDB ID: 5EVM) of the Nipah virus fusion protein obtained from PDB, showing the enlarged position of residue 190.

[Example 2] Gene manipulation based on structural analysis

The proteolytic region of the protein of Example 1 was deleted, and a trimeric motif (foldon) was inserted to enhance protein stability, and Avi-Tag was inserted to confirm protein expression.

Also, a mutant protein (I190P) in which amino acid residue 190 of the HR1 loop is substituted with proline (I190P), a mutant protein in which amino acid residue 114 is substituted with asparagine (I114N), and a double mutant protein of residues 190 and 114 (I190P/I114N) was prepared. Gene mutations were made by site-directed mutagenesis.

[Example 3] Mass production and purification of Nipah virus fusion mutant protein

Nipah virus fusion protein (F0), I190P mutant protein, I114N mutant protein and double mutant (I190P/I114N) protein were respectively produced and purified in insect cells using the Bac-to-Bac expression system.

cell

Sf-9 ( Spodoptera frugiperda 9 ) insect cells were grown in serum-free Sf-900™ II SFM (Gibco BRL, Karlsruhe, Germany) with heat-inactivated 5% FBS and maintained at 27° C. without CO ₂ exchange. . Thereafter, suspension culture was performed at 130 rpm using a shaker in a non-humidified incubator at 26-28 ° C.

High Five (Hi5) cells were grown in serum-free Sf-900™ II SFM and subcultured up to 30 times.

Baculovirus production and protein production

A baculovirus was prepared for mass production of Nipah virus fusion mutant protein, and the produced protein was obtained as follows.

After synthesizing the cDNA of the Nipah virus fusion mutant protein and amplifying it using PCR, it is present under the GP67 secretion signal sequence of the transfer vector pAcGP67A (BD Biosciences, Woburn, MA, USA) pFastBac™ HT A (Invitrogen, Carlsbad, CA). , USA) was cloned into a vector. Then, recombinant bacmid DNA was produced in DH10Bac through a Bac-to-Bac expression system. The produced recombinant bacmid DNA was transfected into Sf-9 cells using Cellfectin II, and the virus was produced after culturing for 2-3 days. After the produced virus was stepwise amplified in Sf-9 cells, Hi5 cells were infected at an MOI of 5 and cultured for 3 days. Thereafter, the cell pellet was removed by centrifugation at 3,000 rpm for 30 minutes and at 10,000 rpm for 20 minutes to obtain a supernatant from which the protein was secreted.

protein purification

Nipah virus fusion mutant protein was isolated and purified from the obtained supernatant using the AKTA BASIC chromatography system as follows.

KTA 정제 시스템(GE HealthCare, Piscataway, NJ, USA)에 연결된 50 mM Tris-HCl(pH 8.0) 및 100 mM NaCl 중 Superdex 200 10/300 GL 컬럼, 또는 20 mM Tris(pH 8.0), 200 mM NaCl 및 10 % 글리세롤 중 Superose 6 증가 컬럼을 사용하여 Size exclusion chromatography (SEC)에 의해 정제하였다. 특성화(characterization)를 위해 Amicon 10,000 MWCO 농축기(Merck Millipore, Billerica, MA, USA)를 사용하여 바이러스 항원 단백질 삼중체를 1-2 mg/ml의 고순도 단백질로 농축시켰다.The supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen, Hilden, Germany) equilibrated with 20 mM Tris-HCl, 100 mM NaCl, pH 8.0. The column is washed with a buffer containing 30 mM imidazole and the protein is eluted in an imidazole gradient (30-400 mM), which is dialyzed against 20 mM Tris-HCl (pH 8.0) and 20-150 mM NaCl. did. Then the protein

Superdex 200 10/300 GL column in 50 mM Tris-HCl (pH 8.0) and 100 mM NaCl connected to a KTA purification system (GE HealthCare, Piscataway, NJ, USA), or 20 mM Tris (pH 8.0), 200 mM NaCl and Purification was made by size exclusion chromatography (SEC) using a Superose 6 augmentation column in 10% glycerol. For characterization, the viral antigen protein triplet was concentrated to a high purity protein of 1-2 mg/ml using an Amicon 10,000 MWCO concentrator (Merck Millipore, Billerica, MA, USA).

3 and 4 show the Ni-NTA affinity chromatography and size exclusion chromatography purification profiles of the Nipah virus fusion double mutant (I190P/I114N) protein. 5 and 6 show the results of SDS-PAGE and Western-blot performed to confirm protein purification.

[Example 4] Confirmation of increased expression of Nipah virus fusion mutant protein

Cell lysates of each of the unmutated Nipah virus fusion protein, I190P mutant protein, I114N mutant protein, and double mutant (I190P/I114N) protein were used to determine whether the expression of Nipah virus fusion protein was increased through genetic manipulation. Protein expression in the supernatant was analyzed.

cell lysate

The transfected cells were lysed in sample buffer (0.1 M Tris, pH 6.8, 4% SDS, 4 mM EDTA, 286 mM 2-mercaptoethanol, 3.2 M glycerol and 0.05% bromophenol blue). Cell lysates were prepared.

Western-blot

Cell lysates and supernatants of Nipah virus fusion protein, I190P mutant protein, and I114N mutant protein were separated by SDS-PAGE, and the protein was detected using an Avi-tag antibody (Genscript, Piscataway, NJ, USA).

Native-PAGE (Native polyacrylamide gel electrophoresis)

Purified double mutant (I190P/I114N) protein was prepared without β-mercaptoethanol and sodium dodecyl sulphate (SDS) in 5x loading buffer, 312.5 mM Tris-HCl (pH 6.8), 50% glycerol. And 0.05% bromophenol blue (bromophenol blue) was prepared under the mixed native conditions. Electrophoresis was performed in 25 mM Tris-HCl buffer (pH 8.8) with 192 mM glycine at 200 V, 80 mA for 1 hour. The gel was stained using Coomassie Brilliant Blue R-250 (Sigma-Aldrich).

As a result of electrophoresis (SDS-PAGE), expression of the Nipah virus fusion protein was not confirmed in both the cell lysate and the supernatant ( FIG. 7 ). On the other hand, the I190P mutant protein was identified as an aggregated form in the cell lysate (FIG. 8), and the I114N mutant protein was expressed in the supernatant (FIG. 9). Also, as shown in FIG. 10 , it was confirmed through Native-PAGE that the Nipah virus fusion double mutant (I190P/I114N) protein was in the form of a hexamer in which two triplex structures were connected.

Through this, it can be seen that the expression of the Nipah virus fusion protein is significantly increased due to the genetic mutation. Therefore, the Nipah virus fusion mutant protein according to the present invention can be used for the development of vaccines and therapeutic agents for preventing or treating Nipah virus infection, and can also be effectively utilized for the development of diagnostic agents based on protein antigens.

<110> Korea Center for Disease Control and Prevention Korea University Research and Business Foundation, Sejong Campus <120> MUTANT FUSION PROTEIN OF NIPAH VIRUS AND ITS USE <130> KCD19P-0007-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 517 <212> PRT <213> Artificial Sequence <220> <223> I190P <400> 1 Ile Leu His Tyr Glu Lys Leu Ser Lys Ile Gly Leu Val Lys Gly Ile 1 5 10 15 Thr Arg Lys Tyr Lys Ile Lys Ser Asn Pro Leu Thr Lys Asp Ile Val 20 25 30 Ile Lys Met Ile Pro Asn Val Ser Asn Met Ser Gln Cys Thr Gly Ser 35 40 45 Val Met Glu Asn Tyr Lys Thr Arg Leu Asn Gly Ile Leu Thr Pro Ile 50 55 60 Lys Gly Ala Leu Glu Ile Tyr Lys Asn Asn Thr His Asp Leu Val Gly 65 70 75 80 Asp Val Arg Leu Ala Gly Val Ile Met Ala Gly Val Ala Ile Gly Ile 85 90 95 Ala Thr Ala Ala Gln Ile Thr Ala Gly Val Ala Leu Tyr Glu Ala Met 100 105 110 Lys Asn Ala Asp Asn Ile Asn Lys Leu Lys Ser Ser Ile Glu Ser Thr 115 120 125 Asn Glu Ala Val Val Lys Leu Gln Glu Thr Ala Glu Lys Thr Val Tyr 130 135 140 Val Leu Thr Ala Leu Gln Asp Tyr Ile Asn Thr Asn Leu Val Pro Thr 145 150 155 160 Ile Asp Lys Pro Ser Cys Lys Gln Thr Glu Leu Ser Leu Asp Leu Ala 165 170 175 Leu Ser Lys Tyr Leu Ser Asp Leu Leu Phe Val Phe Gly Pro Asn Leu 180 185 190 Gln Asp Pro Val Ser Asn Ser Met Thr Ile Gln Ala Ile Ser Gln Ala 195 200 205 Phe Gly Gly Asn Tyr Glu Thr Leu Leu Arg Thr Leu Gly Tyr Ala Thr 210 215 220 Glu Asp Phe Asp Asp Leu Leu Glu Ser Asp Ser Ile Thr Gly Gln Ile 225 230 235 240 Ile Tyr Val Asp Leu Ser Gly Tyr Tyr Ile Ile Val Arg Val Tyr Phe 245 250 255 Pro Ile Leu Thr Glu Ile Gln Gln Ala Tyr Ile Gln Glu Leu Leu Pro 260 265 270 Val Ser Phe Asn Asn Asp Asn Ser Glu Trp Ile Ser Ile Val Pro Asn 275 280 285 Phe Ile Leu Val Arg Asn Thr Leu Ile Ser Asn Ile Glu Ile Gly Phe 290 295 300 Cys Leu Ile Thr Lys Arg Ser Val Ile Cys Asn Gln Asp Tyr Ala Thr 305 310 315 320 Pro Met Thr Asn Asn Met Arg Glu Cys Leu Thr Gly Ser Thr Glu Lys 325 330 335 Cys Pro Arg Glu Leu Val Val Ser Ser His Val Pro Arg Phe Ala Leu 340 345 350 Ser Asn Gly Val Leu Phe Ala Asn Cys Ile Ser Val Thr Cys Gln Cys 355 360 365 Gln Thr Thr Gly Arg Ala Ile Ser Gln Ser Gly Glu Gln Thr Leu Leu 370 375 380 Met Ile Asp Asn Thr Thr Cys Pro Thr Ala Val Leu Gly Asn Val Ile 385 390 395 400 Ile Ser Leu Gly Lys Tyr Leu Gly Ser Val Asn Tyr Asn Ser Glu Gly 405 410 415 Ile Ala Ile Gly Pro Val Phe Thr Asp Lys Val Asp Ile Ser Ser 420 425 430 Gln Ile Ser Ser Met Asn Gln Ser Leu Gln Gln Ser Lys Asp Tyr Ile 435 440 445 Lys Glu Ala Gln Arg Leu Leu Gly Ile His Gly Leu Asn Asp Ile Phe 450 455 460 Glu Ala Gln Lys Ile Glu Trp His Glu Glu Asn Leu Tyr Phe Gln Ser 465 470 475 480 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 485 490 495 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 500 505 510 His His His His His 515 <210> 2 <211> 517 <212> PRT <213> Artificial Sequence <220> <223> I114N <400> 2 Ile Leu His Tyr Glu Lys Leu Ser Lys Ile Gly Leu Val Lys Gly Ile 1 5 10 15 Thr Arg Lys Tyr Lys Ile Lys Ser Asn Pro Leu Thr Lys Asp Ile Val 20 25 30 Ile Lys Met Ile Pro Asn Val Ser Asn Met Ser Gln Cys Thr Gly Ser 35 40 45 Val Met Glu Asn Tyr Lys Thr Arg Leu Asn Gly Ile Leu Thr Pro Ile 50 55 60 Lys Gly Ala Leu Glu Ile Tyr Lys Asn Asn Thr His Asp Leu Val Gly 65 70 75 80 Asp Val Arg Leu Ala Gly Val Asn Met Ala Gly Val Ala Ile Gly Ile 85 90 95 Ala Thr Ala Ala Gln Ile Thr Ala Gly Val Ala Leu Tyr Glu Ala Met 100 105 110 Lys Asn Ala Asp Asn Ile Asn Lys Leu Lys Ser Ser Ile Glu Ser Thr 115 120 125 Asn Glu Ala Val Val Lys Leu Gln Glu Thr Ala Glu Lys Thr Val Tyr 130 135 140 Val Leu Thr Ala Leu Gln Asp Tyr Ile Asn Thr Asn Leu Val Pro Thr 145 150 155 160 Ile Asp Lys Ile Ser Cys Lys Gln Thr Glu Leu Ser Leu Asp Leu Ala 165 170 175 Leu Ser Lys Tyr Leu Ser Asp Leu Leu Phe Val Phe Gly Pro Asn Leu 180 185 190 Gln Asp Pro Val Ser Asn Ser Met Thr Ile Gln Ala Ile Ser Gln Ala 195 200 205 Phe Gly Gly Asn Tyr Glu Thr Leu Leu Arg Thr Leu Gly Tyr Ala Thr 210 215 220 Glu Asp Phe Asp Asp Leu Leu Glu Ser Asp Ser Ile Thr Gly Gln Ile 225 230 235 240 Ile Tyr Val Asp Leu Ser Gly Tyr Tyr Ile Ile Val Arg Val Tyr Phe 245 250 255 Pro Ile Leu Thr Glu Ile Gln Gln Ala Tyr Ile Gln Glu Leu Leu Pro 260 265 270 Val Ser Phe Asn Asn Asp Asn Ser Glu Trp Ile Ser Ile Val Pro Asn 275 280 285 Phe Ile Leu Val Arg Asn Thr Leu Ile Ser Asn Ile Glu Ile Gly Phe 290 295 300 Cys Leu Ile Thr Lys Arg Ser Val Ile Cys Asn Gln Asp Tyr Ala Thr 305 310 315 320 Pro Met Thr Asn Asn Met Arg Glu Cys Leu Thr Gly Ser Thr Glu Lys 325 330 335 Cys Pro Arg Glu Leu Val Val Ser Ser His Val Pro Arg Phe Ala Leu 340 345 350 Ser Asn Gly Val Leu Phe Ala Asn Cys Ile Ser Val Thr Cys Gln Cys 355 360 365 Gln Thr Thr Gly Arg Ala Ile Ser Gln Ser Gly Glu Gln Thr Leu Leu 370 375 380 Met Ile Asp Asn Thr Thr Cys Pro Thr Ala Val Leu Gly Asn Val Ile 385 390 395 400 Ile Ser Leu Gly Lys Tyr Leu Gly Ser Val Asn Tyr Asn Ser Glu Gly 405 410 415 Ile Ala Ile Gly Pro Val Phe Thr Asp Lys Val Asp Ile Ser Ser 420 425 430 Gln Ile Ser Ser Met Asn Gln Ser Leu Gln Gln Ser Lys Asp Tyr Ile 435 440 445 Lys Glu Ala Gln Arg Leu Leu Gly Ile His Gly Leu Asn Asp Ile Phe 450 455 460 Glu Ala Gln Lys Ile Glu Trp His Glu Glu Asn Leu Tyr Phe Gln Ser 465 470 475 480 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 485 490 495 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 500 505 510 His His His His His 515 <210> 3 <211> 517 <212> PRT <213> Artificial Sequence <220> <223> I190P/I114N <400> 3 Ile Leu His Tyr Glu Lys Leu Ser Lys Ile Gly Leu Val Lys Gly Ile 1 5 10 15 Thr Arg Lys Tyr Lys Ile Lys Ser Asn Pro Leu Thr Lys Asp Ile Val 20 25 30 Ile Lys Met Ile Pro Asn Val Ser Asn Met Ser Gln Cys Thr Gly Ser 35 40 45 Val Met Glu Asn Tyr Lys Thr Arg Leu Asn Gly Ile Leu Thr Pro Ile 50 55 60 Lys Gly Ala Leu Glu Ile Tyr Lys Asn Asn Thr His Asp Leu Val Gly 65 70 75 80 Asp Val Arg Leu Ala Gly Val Asn Met Ala Gly Val Ala Ile Gly Ile 85 90 95 Ala Thr Ala Ala Gln Ile Thr Ala Gly Val Ala Leu Tyr Glu Ala Met 100 105 110 Lys Asn Ala Asp Asn Ile Asn Lys Leu Lys Ser Ser Ile Glu Ser Thr 115 120 125 Asn Glu Ala Val Val Lys Leu Gln Glu Thr Ala Glu Lys Thr Val Tyr 130 135 140 Val Leu Thr Ala Leu Gln Asp Tyr Ile Asn Thr Asn Leu Val Pro Thr 145 150 155 160 Ile Asp Lys Pro Ser Cys Lys Gln Thr Glu Leu Ser Leu Asp Leu Ala 165 170 175 Leu Ser Lys Tyr Leu Ser Asp Leu Leu Phe Val Phe Gly Pro Asn Leu 180 185 190 Gln Asp Pro Val Ser Asn Ser Met Thr Ile Gln Ala Ile Ser Gln Ala 195 200 205 Phe Gly Gly Asn Tyr Glu Thr Leu Leu Arg Thr Leu Gly Tyr Ala Thr 210 215 220 Glu Asp Phe Asp Asp Leu Leu Glu Ser Asp Ser Ile Thr Gly Gln Ile 225 230 235 240 Ile Tyr Val Asp Leu Ser Gly Tyr Tyr Ile Ile Val Arg Val Tyr Phe 245 250 255 Pro Ile Leu Thr Glu Ile Gln Gln Ala Tyr Ile Gln Glu Leu Leu Pro 260 265 270 Val Ser Phe Asn Asn Asp Asn Ser Glu Trp Ile Ser Ile Val Pro Asn 275 280 285 Phe Ile Leu Val Arg Asn Thr Leu Ile Ser Asn Ile Glu Ile Gly Phe 290 295 300 Cys Leu Ile Thr Lys Arg Ser Val Ile Cys Asn Gln Asp Tyr Ala Thr 305 310 315 320 Pro Met Thr Asn Asn Met Arg Glu Cys Leu Thr Gly Ser Thr Glu Lys 325 330 335 Cys Pro Arg Glu Leu Val Val Ser Ser His Val Pro Arg Phe Ala Leu 340 345 350 Ser Asn Gly Val Leu Phe Ala Asn Cys Ile Ser Val Thr Cys Gln Cys 355 360 365 Gln Thr Thr Gly Arg Ala Ile Ser Gln Ser Gly Glu Gln Thr Leu Leu 370 375 380 Met Ile Asp Asn Thr Thr Cys Pro Thr Ala Val Leu Gly Asn Val Ile 385 390 395 400 Ile Ser Leu Gly Lys Tyr Leu Gly Ser Val Asn Tyr Asn Ser Glu Gly 405 410 415 Ile Ala Ile Gly Pro Val Phe Thr Asp Lys Val Asp Ile Ser Ser 420 425 430 Gln Ile Ser Ser Met Asn Gln Ser Leu Gln Gln Ser Lys Asp Tyr Ile 435 440 445 Lys Glu Ala Gln Arg Leu Leu Gly Ile His Gly Leu Asn Asp Ile Phe 450 455 460 Glu Ala Gln Lys Ile Glu Trp His Glu Glu Asn Leu Tyr Phe Gln Ser 465 470 475 480 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 485 490 495 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 500 505 510 His His His His His 515

Claims (15)

Consists of the amino acid sequence of SEQ ID NO: 1, Nipah virus fusion mutant protein.
Consists of the amino acid sequence of SEQ ID NO: 2, Nipah virus fusion mutant protein.
Consists of the amino acid sequence of SEQ ID NO: 3, Nipah virus fusion mutant protein.
A gene encoding the protein of any one of claims 1 to 3.
An expression vector comprising a gene encoding the protein of any one of claims 1 to 3.
A host cell transfected with the expression vector of claim 5 .
7. The host cell of claim 6, wherein the host cell is an insect cell.
7. The host cell of claim 6, wherein the host cell is an Sf-9 ( Spodoptera frugiperda 9 ) cell.
preparing an expression vector comprising a gene encoding a Nipah virus fusion mutant protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;
transfecting the expression vector into a host cell;
culturing the transfected host cell to produce a virus;
obtaining a culture by infecting the cells with the virus and culturing; and
Isolating and purifying the protein from the culture
A method for producing a Nipah virus fusion mutant protein comprising a.
The method according to claim 9, wherein the host cell is an insect cell.
The method of claim 9, wherein the host cell is a Sf-9 ( Spodoptera frugiperda 9 ) cell.
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